1. Field of the Invention
The present invention relates to a method for enhancing a binding activity of an antibody composition to Fcγ receptor IIIa, which comprises modifying a complex N-glycoside-linked sugar chain which is bound to the Fc region of an antibody molecule; a method for enhancing an antibody-dependent cell-mediated cytotoxic activity of an antibody composition; a process for producing an antibody composition having an enhanced binding activity to Fcγ receptor IIIa; a method for detecting the ratio of a sugar chain in which fucose is not bound to N-acetylglucosamine in the reducing end in the sugar chain among total complex N-glycoside-linked sugar chains bound to the Fc region in an antibody composition; an Fc fusion protein composition produced by using a cell resistant to a lectin which recognizes a sugar chain in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex N-glycoside-linked sugar chain; and a process for producing the same.
2. Brief Description of the Background Art
Since antibodies have high binding activity, binding specificity and high stability in blood, their applications to diagnosis, prevention and treatment of various human diseases have been attempted [Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 2.1 (1995)]. Also, production of a humanized antibody such as a human chimeric antibody or a human complementarity determining region (hereinafter referred to as “CDR”)-grafted antibody from a non-human animal antibody have been attempted by using genetic recombination techniques. The human chimeric antibody is an antibody in which its antibody variable region (hereinafter referred to as “V region”) is derived from a non-human animal antibody and its constant region (hereinafter referred to as “C region”) is derived from a human antibody. The human CDR-grafted antibody is an antibody in which the CDR of a human antibody is replaced by CDR derived from a non-human animal antibody.
It has been revealed that five classes, namely IgM, IgD, IgG, IgA and IgE, are present in mammal antibodies. Antibodies of human IgG class are mainly used for the diagnosis, prevention and treatment of various human diseases because they have functional characteristics such as long half-life in blood and various effector functions [Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 1 (1995)]. The human IgG class antibody is further classified into the following 4 subclasses: IgG1, IgG2, IgG3 and IgG4. A large number of studies have so far been conducted for antibody-dependent cell-mediated cytotoxic activity (hereinafter referred to as “ADCC activity”) and complement-dependent cytotoxic activity (hereinafter referred to as “CDC activity”) as effector functions of the IgG class antibody, and it has been reported that among antibodies of the human IgG class, the IgG1 subclass has the highest ADCC activity and CDC activity [Chemical Immunology, 65, 88 (1997)]. In view of the above, most of the anti-tumor humanized antibodies, including commercially available Rituxan and Herceptin, which require high effector functions for the expression of their effects, are antibodies of the human IgG1 subclass.
Expression of ADCC activity and CDC activity of the human IgG1 subclass antibodies requires binding of the Fc region of the antibody to a receptor for an antibody (hereinafter referred to as “FcγR”) existing on the surface of effector cells such as killer cells, natural killer cells or activated macrophages and various complement components are bound. Regarding the binding, it has been suggested that several amino acid residues in the hinge region and the second domain of C region (hereinafter referred to as “Cγ2 domain”) of the antibody are important [Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319 (1995), Chemical Immunology, 65, 88 (1997)] and that a sugar chain in the Cγ2 domain [Chemical Immunology, 65, 88 (1997)] is also important.
Regarding the sugar chain, Boyd et al. have examined effects of a sugar chain on the ADCC activity and CDC activity by treating a human CDR-grafted antibody CAMPATH-1H (human IgG1 subclass) produced by a Chinese hamster ovary cell (hereinafter referred to as “CHO cell”) or a mouse myeloma NS0 cell (hereinafter referred to as “NS0 cell”) with various glycosidases, and reported that elimination of sialic acid in the non-reducing end did not have influence upon both activities, but the CDC activity alone was affected by further removal of galactose residue and about 50% of the activity was decreased, and that complete removal of the sugar chain caused disappearance of both activities [Molecular Immunol., 32, 1311 (1995)]. Also, Lifely et al. have analyzed the sugar chain bound to a human CDR-grafted antibody CAMPATH-1H (human IgG1 subclass) which was produced by CHO cell, NS0 cell or rat myeloma Y0 cell (hereinafter referred to as “Y0 cell”), measured its ADCC activity, and reported that the CAMPATH-1H derived from Y0 cell showed the highest ADCC activity, suggesting that N-acetylglucosamine (hereinafter sometimes referred to as “GlcNAc”) at the bisecting position is important for the activity [Glycobiology, 5, 813 (1995); WO99/54342]. These reports indicate that the structure of the sugar chain plays an important role in the effector functions of human antibodies of IgG1 subclass and that it is possible to prepare an antibody having much higher effector function by modifying the structure of the sugar chain. However, structures of sugar chains are various and complex actually, and it cannot be said that an important structure for the effector function was completely identified.
Thus, the sugar chain bound to the CH2 domain of an IgG class antibody has great influence on the induction of effector functions of an antibody. As described above, some of effector functions of an antibody are exerted via interaction with FcγR present on the effector cell surface [Annu. Rev. Immunol., 18, 709 (2000), Annu. Rev. Immunol., 19, 275 (2001)].
It has been found that 3 different types are present in FcγR, and they are respectively called FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). In human, FcγRII and FcγRIII are further classified into FcγRIIa and FcγRIIb, and FcγRIIIa and FcγRIIIb, respectively. FcγR is a membrane protein belonging to the immunoglobulin super family, FcγRII and FcγRIII have an α chain having an extracellular region containing two immunoglobulin-like domains, FcγRI has an α chain having an extracellular region containing three immunoglobulin-like domains, as a constituting component, and the α chain is involved in the IgG binding activity. In addition, FcγRI and FcγRIII have a γ chain or ζ chain as a constituting component which has a signal transduction function in association with the α chain [Annu. Rev. Immunol., 18, 709 (2000), Annu. Rev. Immunol., 19, 275 (2001)].
FcγRI is a high affinity receptor having a binding constant (hereinafter referred to as “KA”) value of 108 to 109 M−1 and also has high binding activity for monomeric IgG [Ann. Hematol., 76, 231 (1998)]. On the other hand, FcγRII and FcγRIII are low affinity receptors which have a low KA value of 105 to 107 M−1 for monomeric IgG and efficiently bind to an IgG immune complex polymerized by binding to an antigen or the like [Ann. Hematol., 76, 231 (1998)]. Based on its functions, FcγR is classified into an activating receptor and an inhibitory receptor [Annu. Rev. Immunol., 19, 275 (2001)].
In the activating receptor, there is a sequence consisting of 19 amino acid residues, called immunoreceptor tyrosine-based activation motif (hereinafter referred to as “ITAM”) in the intracellular region of the α chain or the associating γ chain or ζ chain. According to the binding of an IgG immune complex, a tyrosine kinase such as Src or Syk which interacts with ITAM is activated to induce various activation reactions.
In the inhibitory receptor, there is a sequence consisting of 13 amino acid residues, called immunoreceptor tyrosine-based inhibitory motif (hereinafter referred to as “ITIM”) in the intracellular region of the α chain. When ITIM is phosphorylated via its association with the activating receptor, various reactions including activation of a phosphatase called SHIP are induced to suppress activation signal from the activation receptor.
In human, the high affinity FcγRI and the low affinity FcγRIIa and FcγRIIIa function as activating receptors. In FcγRI, an ITAM sequence is present in the intracellular region of the associated γ chain. FcγRI is expressed on macrophages, monocytes, dendritic cells, neutrophils, eosinophils and the like. FcγRIIa comprises a single α chain, and an ITAM-like sequence is present in the intracellular region. FcγRIIa is expressed on macrophages, mast cells, monocytes, dendritic cells, Langerhans cells, neutrophils, eosinophils, platelets and a part of B cells. FcγRIIIa has an ITAM sequence present in the intracellular region of the associated γ chain or ζ chain and is expressed on NK cells, macrophages, monocytes, mast cells, dendritic cells, Langerhans cells, eosinophils and the like, but is not expressed on neutrophils, B cells and T cells.
On the other hand, the low affinity receptor FcγRIIb comprises a single a chain, and the amino acid sequence in the extracellular region has homology of about 90% with FcγRIIa. However, since an ITMI sequence is present in the intracellular region, it functions as a suppressing receptor. FcγRIIb is expressed on B cells, macrophages, mast cells, monocytes, dendritic cells, Langerhans cells, basophils, neutrophils and eosinophils, but is not expressed on NK cells and T cells. FcγRIIIb comprises a single a chain, and the amino acid sequence in the extracellular region has a homology of about 95% with FcγRIIIa. However, it is specifically expressed on neutrophils as a glycosylphosphatidylinositol (hereinafter referred to as “GPI”)—anchored membrane protein. FcγRIIIb binds to an IgG immune complex but cannot activate cells by itself, and it is considered to function via its association with a receptor having an ITAM sequence such as FcγRIIa. Thus, in vivo effector functions of IgG class antibodies are obtained as the result of complex interaction with activating and suppressing FcγRs expressed on various effector cells.
It is considered that ADCC activity as one of the effector functions of IgG class antibodies is generated as a result of activation of effector cells such as NK cells, neutrophils, monocytes and macrophages, and among these, NK cells play an important role [Blood, 76, 2421 (1990), Trends in Immunol., 22, 633 (2001), Int. Rev. Immunol., 20, 503 (2001)].
FcγR expressed on NK cells is FcγRIIIa. Accordingly, it is considered that the ADCC activity can be enhanced by enhancing the activation signal from FcγRIIIa expressed on the NK cells.
As Fc fusion protein, Etanercept (trade name: Enbrel, manufactured by Immunex) (U.S. Pat. No. 5,605,690) and Alefacept (trade name: Amevive, manufactured by Biogen) (U.S. Pat. No. 5,914,111) and the like are known. Also, it is known that it has no ADCC activity when CH2 domain of an antibody is absent.